Additional file 3 of Nexilin promotes calcium-dependent endo-lysosomal fission required for retrograde transport

Supplementary Material 3: Figure S3. Phosphorylation of nexilin alters myosin II phosphorylation. A U2OS cells transiently transfected with GFP-nexilin wild type (wt), S442E (phosphorylation-mimic mutant), or S442A (phosphorylation-null mutant) were lysed and subjected to Western blot analysis using antibodies against phosphorylated myosin light chain on Ser 19 (pMLC), total myosin light chain (MLC), nexilin and tubulin. B Quantification of the levels of phosphorylated myosin light chain on Ser 19 (pMLC). The intensity of the bands from the Western blots were measured using ImageJ and normalised to the total myosin light chain (MLC) levels, using tubulin as loading control. Data represents the mean ± s.d. from three independent experiments. One-way ANOVA followed by Tukey’s post-hoc test was applied for statistical analysis. C U2OS cells transiently transfected with mCherry-LAMP1 were treated with 20µM ML-SA1 for 1 hour, or left untreated, then incubated with 2µM LysoSensor Yellow/Blue DND-160 for 10 minutes before fixation and imaging Scale bar: 10 µm. ns non-significant, * p < 0.05, ** p < 0.01